A central component of host defence systems against invading bacterial, fungal, yeast and viral pathogens involves the successful recognition of the pathogen, or components thereof, by cellular receptors which induce a signalling cascade resulting in stimulation of the immune system and the production of various cytokines. One family of receptors which plays a key role in this line of defence is the Toll-like receptor (TLR) family. TLRs are expressed on immune cells including monocytes, macrophages, dendritic cells, B cells and granulocytes, and a variety of other cell types including endothelium and epithelium. TLR activation by pathogens, or by molecules derived therefrom, induces intracellular signalling that primarily results in activation of the transcription factor NF-κB (Beg, 2002, Trends Immunol 23:509-12.) and modulation of cytokine production. However, a series of other pathways can also be triggered, including p38 mitogen activated kinase, c-Jun-N-terminal kinase and extracellular signal related kinase pathways (Flohe et al., 2003, J Immunol 170:2340-2348; Triantafilou and Triantafilou, 2002, Trends Immunol 23:301-304).
Type I interferons, principally IFNα and IFNβ, are major contributors in the immune response to bacterial and viral infections. The primary source of type I interferons following such infections are plasmacytoid dendritic cells (PDCs), a specialised subpopulation of peripheral blood mononuclear cells. PDCs express the Toll-like receptors TLR7 and TLR9 and it is TLR7/TLR9 stimulation that is responsible for PDC activation and the production of large quantities of type I interferons.
A range of pathogen-derived factors are capable of selectively stimulating different TLRs. One of the best characterised of these is lipopolysaccharide (LPS), a glycolipid derived from the outer membrane of Gram-negative bacteria, which is an agonist of TLR4. TLR2 recognizes lipoteichoic acid (LTA), petidoglycan (PGN) and lipopeptide, TLR3 recognises double-stranded RNA, typically of viral origin; TLR 7 recognises viral single-stranded RNA and TLR9 recognises unmethylated CpG dinucleotides, typically within bacterial and viral DNA. TLRs can also be stimulated by a variety of synthetic agonists, for example polyI:C (polyinosinic-polysytidilic acid) in the case of TLR3, imidazoquinolines such as resiquimod (R848) and imiquimod in the case of TLR7 and CpG-rich oligonucleotides in the case of TLR9.
The elucidation of mechanisms for the modulation of TLR signalling and modulation of TLR-stimulated cytokine and chemokine production will provide avenues for the development of therapeutic approaches to the treatment of a variety of diseases and conditions, including viral and bacterial diseases.
Mammalian chaperonin 10 (Cpn10), also known as heat shock protein 10 (Hsp 10), is typically characterised as a mitochondrial ‘molecular chaperone’ protein involved in protein folding together with chaperonin 60 (Cpn60; Hsp60). Cpn10 is a homologue of the bacterial protein GroES. GroES and Cpn10 oligomerise into seven member rings that bind as a lid onto a barrel-like structure that comprises fourteen GroEL or seven Hsp60 molecules, which tether denatured proteins to the complex. Cpn10 is also frequently found at the cell surface (Belles et al, 1999, Infect Immun 67:4191-4200) and in the extracellular fluid (Shin et al, 2003, J Biol Chem 278: 7607-7616).
However Cpn10 has also been shown to possess immunosuppressive activity in experimental autoimmune encephalomyelitis, delayed type hypersensitivity and allograft rejection models (Zhang et al., 2003, J Neurol Sci 212:37-46; Morton et al., 2000, Immunol Cell Biol 78:603-607).
Previously, the present inventors have also demonstrated that in the presence of TLR4 and TLR2 agonists (LPS and PAM3CysSK4, respectively), Cpn10 reduces TLR4- and TLR2-stimulated NF-κB activation, reduces TNF-α and RANTES secretion, whilst increasing IL-10 secretion in a dose-dependent manner (Johnson et al., 2005, J Biol Chem 280:4037-4047; International Patent Application No. PCT/AU2005/000041; WO2005/067959, the disclosures of which are incorporated herein by reference).
The inventors have now surprisingly found that Cpn10 also modulates signalling by TLR3, TLR7 and TLR9. Cpn10 is shown herein to dose-responsively enhance the production of IFNα and IFNβ in the presence of a TLR3-specific ligand, and to reduce NF-kB activation in the presence of TLR7- and TLR9-specific ligands. Moreover, the inventors have surprisingly shown that Cpn10, but not Cpn60 or GroES, associates with TLRs in activation clusters at the cell surface, with such activation clusters modulating TLR signalling.